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Jan 21, 2016 - traits, essential oil composition and DNA markers is useful in verifying the taxonomy of Ocimum. Keywords: Plant Conservation, Genetic ...

Available online on www.ijppr.com International Journal of Pharmacognosy and Phytochemical Research 2016; 8(2); 252-262 ISSN: 0975-4873 Review Article

Diversity Analyses in Ocimum Species: Why and How? Sarwat M1*, Srivastava S1, Naved T 1

Pharmaceutical Biotechnology, Amity Institute of Pharmacy, Amity University, NOIDA, Uttar Pradesh 201303. 2 Amity Institute of Pharmacy, Amity University, NOIDA, Uttar Pradesh 201303. Available Online:21st January, 2016

ABSTRACT Basil is an important part of Lamiaceace family. Besides having various economical importance it also has diverse uses in the traditional medicines. It is rich in camphor, geraniol, linalool and linalyl acetate. The genus is highly variable and possesses wide range of diversity at the level of morphology, chemical and genetic make-up. For analyzing the variation existing in the genus Ocimum, the chemical analyses of its essential oils and DNA fingerprinting were prominently utilized. This is utmost helpful in plant improvement programs as well as developing a well-organized way to conserve the genetic wealth of the genus. In this paper, we have thrown light on how the combinatorial approach of studying the morphological traits, essential oil composition and DNA markers is useful in verifying the taxonomy of Ocimum. Keywords: Plant Conservation, Genetic diversity, Ocimum spp., Authentication, Molecular Markers, DNA Fingerprinting INTRODUCTION Genus Ocimum contains more than 150 species, collectively called as 'Basil'. It comprises annual and perennial herbs and shrubs of Asia, Africa, Central and South America1. In India, its considered as a sacred plant and its leaves are routinely used in worship. Here, three main forms of 'Tulsi' are: 'Rama' or green tulsi, 'Krishna' or purple tulsi and 'Vana' tulsi. Besides being a rich source of essential oils and aromatic compounds2, it is also valued as a culinary herb and an attractive and fragrant ornamental plant3. It is inhabitant of moist soils throughout the world4. Depending on the variations in the soil type and rainfall the size and form of the plants may vary and correspondingly their medicinal strength and efficacy. American basil is a high quality sweet basil with violet leaves of uniform size. While, Egyptian and African Basil have different taste but have lower value in comparison to French and American Basil5. Lemon basil is grown as fresh culinary herb and dried in floral arrangements and is a most widely used variety in drug, perfume and cosmetic industry6. The other species of Ocimum like O. africanum Lour.(syn. O. 9 citriodorum Vis.), O. americanum L. (syn. O. canum Sims.),O. basilicum L.,O. gratissimum L.,O. minimum L., and O. tenuiflorum L. (syn. O. sanctum L.) are also preferred to be grown due to their high medicinal and economic importance. O. kilimandscharicum Baker ex Gu¨rke is known for camphor production, O. gratissimum L of essential oil, O. tenuiflorum L. and O. campechianum Mill. for ornamental and medicinal importance1. Table 1 summarizes different species of Ocimum along with their medicinal value, secondary metabolites and geographical regions. Essential Oil Contents of Ocimum; its Economic Importance

*Author for Correspondence

Several chemotypes of Ocimum has been known, based on the composition of their essential oil2. It is the genetic make-up of the plant which decides its chemical composition and in turn determines its aroma7,8. The oil consist of eugenol, eugenal, carvaled, methyl chavicol, limatrol and caryophylline and a number of sesquiterpenes. and monoterpenes viz., barnyl acetate, β-elemense, methylengenol, neral, β-pinene, comphene, α-pinene etc. They have been used as antifungal9,10, antioxidant11,12, antinociceptive13, anticonvulsant14, germicidal15,16 and antimalarial17. Problem of Adulteration and Substitution of the Herbal Drugs The herbal drug industry is facing a major problem of adulteration and substitution of herbal drugs. Thus, it's a matter of concern while dealing with research on commercial natural products18. The factors responsible for adulteration are lack of knowledge of authentic sources, similarity in colour and morphology, careless collection by herbal collectors and suppliers, non availability of native drug and sometimes high cultivation cost of these drugs in wild19. In the case of Ocimum sanctum, its often found adulterated with its morphologically close relative Vitex negundo19. The latter plant has same morphological characters like leaf and flower colour, size and shape (Figure 1). To resolve this problem of misidentification and adulteration various techniques have been utilized since time immemorial, which are described in this review along with their advantages and disadvantages. Classification and Taxonomy of Ocimum Ocimum species have high levels of phenomic, genetic and metabolic diversity. The cause of this high level of morphological and chemical variability is attributed to inter-specific and intra-specific hybridization, polyploidy

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and evolutionary selection. Humans have influenced the selection, cultivation, and hybridization of the genus, making it difficult for the taxonomists to work upon. Classification of Ocimum Based on Cytology Ocimum possess two basic chromosome numbers X = 8 and X =12 (20, 21). Species belonging to "sanctum" group have chromosome number X = 8. The chromosomes of Ocimum can be divided into three groups: Group A: Long; 300 microns Group B: Medium; between 2.0 to 2.5 microns Group C: Short; 1.5 microns and below It is assumed that the evolution of closely related species of Ocimum has been through structural differences and repatterning of chromosomes. There appears to be a general reduction in total length in chromatin during evolution22,23. Classification of Ocimum Based on Morphological Characters The taxonomy of Basil genome is considered to be vast and complex. More than 150 species were recognized in the genus24. Actually, most of the taxa described by them was based on the morphology and colour of leaves, which frequently depends on environmental conditions leading to ambiguity in the classification within the genus. There's enormous variation in the shape and colour of the leaves within Ocimum basilicum and its close relatives. The shape varies from small and liniform to large and round and colour varies from yellow-green to grey-green, to red or to almost black. In view of this, it has been proposed that only 65 species of the genus Ocimum should be considered as true species, and the other should be discarded as their synonyms or false attribution1. Ibrahim et al.4 carried out study on seeds of three varieties i.e, French, Purple and Lemon basils, during two growing seasons of 2010 and 2011 at South Tahrir Agricultural Company, Beheira Governorate, Egypt. The results of the study revealed that phenotypic coefficient of variation (PCV), genotypic coefficient of variation (GCV), heritability (HB) and expected genetic advance (GA %) had the highest values in case of herb dry weight, stem dry weight, leaf dry weight and linear growth, respectively. The lowest values of these items were essential oil percent and number of primary branches. In another study done by Carovic Stanko et al.25 on the morphological characters of Ocimum during the year 2006 in the field trial, exhibit twenty-three morphological traits to be polymorphic out of the twenty-seven traits studied among all the accessions. The monomorphic traits observed were: colour of style, number of flowering shoots, serration of leaf blade margin and hairiness of bracts. Average phenotypic dissimilarity was 0.551 between all pairs of accessions. Classification of Ocimum Based on Biochemical Characters Further advancement in the classification of Ocimum was based on the volatile oil composition26. It uses the most prevalent aromatic compounds for classifying the different basil chemotypes. The components which are more than 20%, are taken into consideration. Essential oils varies with the cultivar type, the prevalent components are monotherpenes and phenylpropanoids. Many Ocimum

species contain primarily monoterpene derivatives such as limonene, camphor, 1,8-cineole, linalool and geraniol. Others, including O. basilicum, contain primarily phenoyl derivatives, such as eugenol, methyleugenol, chavicol, estragole, methyl-cinnamate, often combined with various amounts of linalool. Labra et al.27 studied nine O. basilicum accessions most commonly utilized in the Mediterranean area for culinary and ornamental use for chemical analysis. As expected, the results showed environmental influence on the morphological and chemical traits. Previous studies28,29 on these cultivars showed the influence of growth stage in the composition of aromatic compounds. Johnson et al.30 have shown how the light regime can also influence the essential oil compositions within the same genotype. Miele et al.28 have reported that some chemical constituents are purely dependent on external characters of plants; e.g. eugenol and methyleugenol content is strictly related with plant height: methyleugenol is predominant in plants up to 10 cm height, while eugenol is prevalent in taller plants. Labra et al.27 analysed nine O. basilicum accessions through GC/MS analysis and found substantial variation in their chemical composition. Among them, the most prominent constituent was linalool, ranged between 19 and 38%. Other components reported to be present in all the cultivars were eugenol, cineole, terpineol and farnesene Terpineol and farnesene were detected, although at a lower concentration. On the other hand, when De Masi et al.7 analyzed 12 different accessions corresponding to nine different cultivars of O. basilicum, they found the prevalent chemical components as phenylpropanoids (estragole, eugenol, methyl-eugenol and methyl-cinnamate) and monoterpenes (linalool, geranial, neral and eucalyptol) in all 40 different components of essential oils. Based on the study De Masi et al.7 grouped these accessions into five cultivar types. In this way, European or sweet basil oil, from Bulgaria, Egypt, France, Hungary, Italy, South Africa and occasionally in the USA, matches with types I and II, because it contains almost identical amounts of estragole (20–43%) and linalool (37–55%). Exotic or R´eunion basil oil, comprising high estragole content (75–85%, estragole type), does not correspond to the accessions in study. Other two distinct types of oils are linalool type (linalool eugenol) and methyl-cinnamate type (methyl-cinnamate linalool), which are related to types III and IV, respectively. The recent studies by Javanmardi et al.29 and Rady and Nazif31 revealed that rosmarinic acid is the predominant phenolic acid present in both flower and leaf tissues of Ocimum. Hence, the conclusion drawn from these studies is that the chemotype classification based on just one major volatile oil is erroneous, because, one plant may contain two or more chemical compounds in nearly equal amounts. The overall oil profile of major constituents above a fixed threshold level (e.g. 20% of total essential oil content) should be taken into account. Classification of Ocimum based on Molecular Markers

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1.

2.

3.

4.

5. 6.

7. 8.

9.

Table 1. Diversity in the Secondary Metabolite content and Medicinal Importance of Ocimum spp. S.No. Species and cultivars Medicinal Secondary Geographical Importance Metabolite location Ocimum sanctum antispasmodic, Eugenol, ursolic All regions of India antiasthamatic, expectorant, acid, carvacol and hepatoprotective, rosmarinic acid antipyretic Ocimum basilicum stimulant, antispasmodic, Phenolic acid and Cultivated diuretic, demulcent rosmarinic acid throughout India mainly in Punjab Ocimum basilicum stimulant, antispasmodic, estragol Cultivated Var. Thai Basilikum diuretic, demulcent throughout India mainly in Punjab Ocimum Neuralgia, cephalalgia Eugenol Northern India, gratissimum and antifertility FlavonBrazil, China, Cirsimaritin Eastern Nepal and Southern India Ocimum africanum Estragol, geranial, Tropical parts of neral, thymol Africa Ocimum tenuiflorum expectorant, analgesic, 1,8 cineole, beta Northern India anticancer, antiasthmatic, bisabolene antiemetic, diaphoretic, antidiabetic, antifertility, hepatoprotective, hypotensive, hypolipidmic Ocimum Febrifuge, hypoglycaemic, Geraniol, neral, Lower hills of India americanum leucorrhea and antifungal Rosmarinic acid Ocimum Insecticidal, mosquito Camphor, Himalayan region killimandscharicum repellent, spasmolytic and thymol/p–cimene antibacterial Ocimum Antibacterial, antifertility Thymol Northern India, macrophyllum FlavonBrazil, China, Xantomicrol Eastern Nepal and Southern India

Reference 54

55

8

56, 57

8 8

31 8

57

10. Molecular, or DNA-based markers have been utilized for medicinal plant studies since time immemorial and have advantages over other conventional methods like HPLC, GC-MS, Gas Chromatography, TLC etc18,32. They are the most recent to be developed. The primary advantages of using molecular markers are: (1) availability of unlimited number of markers; and (2) generally unaffected by developmental differences or environmental influences. The two general classes of molecular markers used for such studies are DNA-DNA hybridization and polymerase chain reaction (PCR) based. Those based on the Restriction fragment length polymorphism (RFLP) markers depend on DNA-DNA hybridization and have been used for organelle genetic analysis and genetic linkage mapping in forest trees. These DNA based markers have been utilised extensively for studying genetic relationship in different species of Ocimum, as summarised in Table 2. They are also proved useful in differentiating various cultivars or varieties of a particular spp. e.g. O. basilicum (Table 3). Some of these examples are explained in the forthcoming sections. Molecular Biological Analyses of Genus Ocimum The Molecular Biological studies on the genus Ocimumare summarized here. RAPD Analyses of Ocimum species

Randomly amplified polymorphic DNA (RAPD) markers are developed by PCR of anonymous DNA sequences from random primers (generally decamers)33,34,35. These are dominant markers, i.e. The heterozygous (1 copy) and homozygous (2 copies) bands are indistinguishable. Inspite of limitations, the RAPD analysis has emerged as a powerful technique for detecting DNA polymorphisms among cultivars or clones belonging to plant kingdom28,32,36. Some of the drawbacks of RAPD technology can be overcomed by careful planning of experiments. Different workers have given different views on the co-migrating RAPD bands. Some said they might not be allelic i.e. composed of similar sequences37, while some others demonstrated the homology of these comigrating RAPD bands36,38. The use of large number of polymorphic markers minimize the skewing of results due to non-allelism39. Another problem often encountered and questioned regarding RAPD analysis is the reproducibility of banding pattern. This problem can be resolved by thoroughly optimising the PCR reaction conditions and following the same protocol each time. For more accurate analysis, reaction should be performed twice or thrice, scoring only those bands that are reproducible in each reaction32,35.

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Singh et al.40 have utilized RAPD markers and found it to be a robust and reliable method to detect intra- and interspecific genetic diversity. They have studied phylogenetic relationship in the 32 accessions belonging to five Ocimum species and detected high degree of polymorphism (98.28%). The dendrogram constructed grouped three species, namely, O. basilicum, O. kilimandscharicum and

O. americanum in Section A and the other two species O. tenuiflorum and O. gratissimum in Section B. Accessions from one species were clustered together within the Intracluster as expected. The study proved RAPD technique to be sensitive, precise and efficient for phylogenetic studies in the genus Ocimum.

Table 2 : Molecular Markers Used for Analyses of Polymorphism in Ocimum spps: S. Name of species of Place of Type of %polymorphism No. Ocimum and its collection Marker used or % similarity cultivars 1. O. sanctum New Vallabh RAPD , 100% Vidyanagar, ISSR and polymorphic Anand, Gujarat SSR bands Bangalore, India RAPD 13.84 to 3.07% polymorphism Chitrakoot and RAPD 92.31% Bhopal., Madhya polymorphism Pradesh, India 2. O. basilicum University of AFLP 44.7% Zagreb, Croatia polymorphism Anand, Gujarat, India Taiwan

CIMAP, Lucknow, India

RAPD , ISSR, SSR ISSR, RAPD, SRAP RAPD RAPD RAPD

3.

4.

5.

O. viride

O. polystachyon

O. americanum

O. gratissimum

References

Genetic diversity

47

Genetic diversity

23

Interspecies association

58

Genetic diversity

8

100% polymorphism 97% polymorphism

Genetic diversity

47

Genetic diversity

51

98.20% Polymorphism 13.84 to 3.07% polymorphism 92.31% polymorphism 44.7% polymorphism

Interspecific diversity Genetic diversity

41

Interspecies association Interspecific diversity

58

23

University of Zagreb, Croatia

AFLP

Anand, Gujarat India University of Zagreb, Croatia

RAPD , ISSR, SSR AFLP

100% polymorphism 44.7% polymorphism

Genetic diversity

47

Interspecific diversity

8

Anand Agricultural University (AAU), Anand, Gujarat University of Zagreb, Croatia Anand, Gujarat, India Taiwan

RAPD , ISSR, SSR

100% polymorphism

Genetic diversity

47

AFLP

44.7% polymorphism 100% polymorphism 97% polymorphism 98.20 % polymorphism 89% similarity

Interspecific diversity Genetic diversity

8

Genetic diversity

52

Interspecific diversity Interspecies relationship Interspecific diversity

40

CIMAP, Lucknow, India Southern India 6.

Purpose of the Study

University of Zagreb, Croatia

RAPD , ISSR, SSR RAPD, SRAP, ISSR RAPD RAPD AFLP

44.7% polymorphism

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Anand, Gujarat, India

RAPD , ISSR, SSR

Taiwan

RAPD, ISSR,SRAP RAPD

CIMAP, Lucknow, India

7.

O. killimandscharicum

8.

O. campechianum

9.

O. africanum

10.

O. tenuiflorum

Southern India

RAPD

Bangalore, India

RAPD

Chitrakoot and Bhopal, Madhya Pradesh, India University of Zagreb, Croatia CIMAP, Lucknow, India Southern India

RAPD

Bangalore, India

RAPD

Chitrakoot and Bhopal, Madhya Pradesh, India University of Zagreb, Croatia Southern India University of Zagreb, Croatia University of Zagreb, Croatia

RAPD

Taiwan

SRAP, RAPD, ISSR RAPD

CIMAP, Lucknow, India Southern India 11.

O. micranthum

12.

O. canum

Southern India Chitrakoot and Bhopal, Madhya Pradesh, India Chitrakoot and Bhopal, Madhya Pradesh, India

AFLP RAPD RAPD

AFLP RAPD AFLP AFLP

RAPD

100 % polymorphic bands 97% polymorphic bands 98.20% polymorphic bands 89% similarity 13.84 to 3.07% polymorphism 92.31% polymorphism 44.7% polymorphism 98.20% polymorphism 89% similarity 13.84 to 3.07% polymorphism 92.31% polymorphism 44.7% polymorphism 89% similarity 44.7% polymorphism 44.7% polymorphism

97% polymorphism 98.20% polymorphism 89% similarity

Genetic diversity

47

Genetic diversity

51

Interspecific genetic diversity

40

Interspecies relationship Genetic diversity

42

Genetic diversity

58

Interspecific diversity Genetic diversity

8

Interspecies relationship Genetic diversity

43

Genetic diversity

58

Interspecific diversity Genetic diversity Interspecific diversity Molecular and chemical characterization, genetic diversity Genetic diversity

8

Genetic diversity

40 43

58

RAPD RAPD

89% similarity 92.31% polymorphism

Interspecies relationship Genetic diversity Interspecies association

RAPD

92.31% polymorphism

Inter species association

23

40

23

43 8 8

51

43 58

Table 3: Analyses of Intraspecific Genetic Diversity in Ocimum basilicum cultivars/varieties Through Molecular Markers S. Variety Cultivar and its origin Types of % polymorphism/% Reference No. Marker similarity 1. O. basilicum var. Genovese (Austria, AFLP 66.7% polymorphism 8 basilicum Macedonia, Croatia, RAPD 57.5% polymorphism 7 Italy and Slovakia) AFLP 50.9% polymorphism 27 similarity index: 0 to 0.18 AFLP, RAPD 93% polymorphism 59

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RAPD, ISSR, SRAP Sweet USA)

2. 3

4.

basil(Croatia,

AFLP AFLP, RAPD RAPD RAPD, ISSR, SRAP

Green Gate

AFLP

Canada

RAPD, ISSR, SRAP

Grosses gruenes Oriental basil Green globe Canada

AFLP AFLP AFLP RAPD, ISSR, SRAP

O. basilicum var. difforme O. basilicum var. thyrsiflorum

Napoletano Basil Difforme Benth (Italy) Thai- Basilikum Canada

AFLP AFLP RAPD AFLP RAPD, AFLP,SRAP

O. basilicum var. purpurascens

Benth Mexican Basil Anise Basil

RAPD, AFLP AFLP AFLP RAPD, ISSR, SRAP

Cinnamon Basil Dark Opal (German, Russia Slovakia Canada

5.

O. basilicum var. minimum

Rubin Basil Bush Basil (Italy) Canada

AFLP RAPD AFLP RAPD, AFLP ISSR,RAPD, SRAP

RAPD AFLP AFLP RAPD, ISSR, SRAP

Folgia di Lattuga (Italy)

RAPD RAPD

Lemon Basil(Canada)

RAPD

RAPD: 95% polymorphism, ISSR: 97%, SRAP: 93% polymorhism 66.7% polymorhism 93% polymorphism 44.83% polymorphism RAPD: 95% polymorphism, ISSR: 97%, SRAP: 93% polymorhism 85.18% polymorphism

51

RAPD: 95% polymorphism, ISSR markers showed 97% and SRAP markers showed 93% polymorhism 85.18% polymorphism 85.18% polymorphism 66.7% polymorphism RAPD markers showed 95% polymorphism, ISSR: 97%, SRAP: 93% polymorhism 85.18% polymorphism 85.18% polymorphism 93% polymorphism 66.78% polymorphism RAPD: 95% polymorphism, ISSR: 97% and SRAP: 93% polymorhism 93% polymorphism 85.18% polymorphism 85.18% polymorphism

51

RAPD: 95% polymorphism, ISSR: 97% and SRAP: 93% polymorphism 85.18% polymorphism 57.5% polymorphism 66.7% polymorphism

52

93% polymorphism RAPD: 95% polymorphism, ISSR: 97% and SRAP: 93% polymorhism 57.5% polymorphism 85.18% polymorphism 85.18% polymorphism RAPD: 95% polymorphism, ISSR: 97% and SRAP: 93% polymorhism 57.5% polymorphism 50.9% polymorphism; similarity index: 0 to 0.18 57.5% polymorphism

59 51

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25 25 8 51

25 25 59 8 51

59 25 25

25 7 8

7 25 25 41

7 27 7

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RAPD, ISSR, SRAP

a

RAPD: 95% polymorphism, ISSR: 97% and SRAP: 93% polymorphism

51

b

Figure 1: Similarity in morphological features of two different plants (a) Ocimum sanctum and (b) Vitex negundo

In the previous reports of diversity analyses eleven species of Ocimum have been divided into two groups‘Basilicum’ and ‘Sanctum’ on the basis of their morphology, cytology and oil charactersistics22,41. O. basilicum, O. americanum and O. kilimandscharicum were grouped as ‘Basilicum’, while O. tenuiflorum and O. gratissimum were grouped as ‘Sanctum’. Paton1 also placed O. basilicum with O. americanum and O. kilimandscharicum in one sub-section (Ocimum subsect. Ocimum). This shows, that the use of molecular markers helps in more objective classification. Chikkaswamy et al.23 revealed the genetic relationships between six Ocimum species (two O. basilicum, two O. sanctum, one O. kilimandscharicum, one O. gratissimum) (Table 1). Of the 80 random primers screened 10 primers resulted in 73 RAPD bands of good amplification. The data structure included a total of 100 to 1000 base pairs marker levels. A dendrogram was constructed using Euclidean distances by Ward’s method. Based on number of bands all the species were grouped into three clusters and the dendrogram maximum similarity between the Ocimum species. The highest distance was observed between O. killimandscharicum and O. sanctum and lowest distance was recorded between O. basilicum 1 and O. basilicum 2 and intermediate distance was observed between O. sanctum 1 and O. sanctum 2. From the pattern of clustering, it was pertinent that RAPD technique was efficient in segregating species into different clusters. In another study by Harisaranraj et al.42 genetic relationship of seven species was estimated using 15 randomly amplified polymorphic DNA primers. It shows 89% close similarity of O. basilicum with O. tenuiflorum , O. gratissimum and O. micranthum. The results are

surprising, but cannot be trusted upon as the data is based on only two primers. As, other thirteen primers utilised by these workers produced monomorphic bands across all the seven species. De Masi et al.7 have collected 12 different accessions from nine different cultivars. RAPD analysis grouped them into two main clusters. One cluster comprised of cultivars which are suitable for food industry. Interestingly, the results of agro-morphological analysis matches with those of RAPD, but, the essential oil profiles differ from both of these results. Maybe due to difference in the key enzymes of the essential oil biosynthetic pathways. These reports prove that DNA-based markers may represent a routine tool to verify the identity and the quality of basil cultivars and basil-derived products. AFLP Analyses of Ocimum species This technique involves digestion of total cellular DNA with one or more restriction enzymes and ligation of adaptors to the sticky ends. This is followed by amplification of these fragments using two PCR primers that have sequences complimentary to the adaptors and restriction site43. The PCR products are finally resolved on PAGE matrix and visualized through autoradiography or chemiluminescence. There are various advantages of AFLP over other genetic markers; like high reproducibility, resolution, sensitivity at whole genome level, and no need of prior sequence information for amplification. AFLP also has the capability to amplify between 50 and 100 fragments at a time32,44. Carovic- Stanko et al.8 have studied DNA fingerprinting and chemical analysis of essential oils in 22 Ocimum accessions belonging to five different species, viz., O. basilicum, O. americanum/O. africanum, O. tenuiflorum, and O. gratissimum. The chemical analysis identified 17 compounds as the main ones, and thus grouping the 22

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accessions into 13 chemotypes. Whereas, AFLP data has further resolved the relationship between these accessions. 1,734 polymorphic bands were generated by Four AFLP primer combinations. The Dice’s distance matrix and maximum parsimony (MP) analysis grouped these accessions into four clusters. The O. campechianum accession was not able to group together with other species, also proved by its unique essential oil composition with 1,8-cineole and b-caryophyllene as the main compounds. O. gratissimum and O. tenuiflorum are also separated out from other accessions. This is in congruence with the studies of Khosla41, where both of them were placed in Sanctum group. The study also substantiated by the smaller number of chromosomes possess by O. gratissimum (2n = 40) and O. tenuiflorum (2n = 36) in comparison of O. basilicum (2n = 48) and O. americanum (2n = 72) (25). Another study on nine accessions of Ocimum basilicum revealed 163 bands, 83 of which were polymorphic 27. The accessions B and D show high genetic similarity with C and G. While, accessions A, H, I, E, and F are clearly separated from each other and from the other accessions, the accessions B, C, D, and G exhibit no genetic variability among individuals within the cultivar, suggesting them to be clones. The dendrograms exhibit low genetic variability thus showing closely related species. It may be because of autogamous reproduction and propagation in O. basilicum. The results suggest that it is not always necessary that the genomic similarity coincide with other morphological and physiological traits, such as oil composition, or agronomic traits etc. For example, although, cultivars B and D are quite different in their oil composition but they are genetically very similar. ISSR and SSR Marker Analyses of Ocimum species PCR-based techniques are much preferred among all the marker techniques because of their simplicity and requirement of small quantities of DNA template18,32. Nonanchored inter simple sequence repeats (ISSRs) are one of the PCR based markers dependendent on microsatellite loci. Like RAPD markers, they also do not require any prior genomic information45. The ISSR technique has one major advantage of its consistency across a wide range of PCR parameters46. In a study by Lal et al.47 RAPD, ISSR and SSR markers for studying genetic relationship among six different species of Ocimum. Out of the total 209 bands amplified by three ISSR, all were polymorphic. Analysis of all three markers also showed 100% polymorphism among the Ocimum spp. RAPD and SSR analyses, revealed that O. basillicum and O. polystachyon are most similar (highest similarity index of 0.28000 and 0.61538, respectively). Through ISSR analysis, the highest similarity index of 0.32258 was observed between O. americanum and O. basillicum. The combined analysis of RAPD, ISSR and SSR revealed that O. basillicum and O. americanum were the most similar (highest similarity index is 0.2892) and O. viride and O. americanum are highly dissimlar (least similarity index is 0.01123). Information from all the three marker systems can be utilised to formulate a strategy for plant breeding

and crop improvement programs by maintaining or enhancing the genetic diversity. SRAP Marker Analyses of Ocimum species Sequence-related amplified polymorphism (SRAP) is a simple and reliable method with moderate throughput ratios48, 49. It amplifies the coding sequences in the genome and results in a moderate number of co-dominant markers. Careful analysis of sequence data revealed that codominant markers are generated from fragment size changes due to insertions and deletions, wheras, dominant markers are generated from nucleotide changes. The SRAP marker system has been utilised for a variety of purposes like genetic diversity studies, map construction, gene tagging etc50. SRAP analysis of 37 basil accessions representing four Ocimum species (Ocimum basilicum, O. americanum, O.gratissimum and O.tenuiflorum, revealed highest mean value of polymorphic information content (PIC, 0.29) and resolving power (Rp, 30.19) which was much higher than those of RAPD (0.23, 5.13) and ISSR (0.19, 1.39)51. When ISSR and RAPD markers were used, all the accessions were clustered into four groups, but SRAP analysis grouped them into three clusters, indicating that the genetic diversity in different target regions of the tested basil genome was not the same. The SRAP dendrogram was correlated most with the combined data set indicating that SRAP markers could be better tool for genetic diversity analysis in basil than RAPD and ISSR markers 51. DNA Barcoding of Ocimum species DNA barcodes is the latest technique in DNA fingerprinting. It utilises the genome of mitochondria, chloroplast and nuclear regions and proved to be of great success in the validation of several plant families52,53. In the genus Ocimum, the three candidate barcodes of the chloroplast genome viz. matK, rbcL and psbA-trnH were analysed to access their ability to produce high sequence variability. The sequencing data revealed psbA-trnH region as the most suitable candidate barcode which presented an interspecific variation of 7.3%. Clear differentiation observed at the species level represent the maximum distance to be 0.264 among the dissimilar species. The phylogenetic analysis have identified the hybrids too. The DNA barcoding is proved to be ideal for interspecific differentiation of Ocimum. CONCLUSIONS The DNA based marker technologies are useful in assigning new unclassified accessions to pre-existing taxonomic groups and also rectifying any mistakes in the classification done by more primitive methods, like phenotyping and chemotyping. Hence, it is a more objective approach for phylogenetic studies. The newest technologies in the field of DNA genotyping, viz., DNA barcoding, Microarrays and Next Generation Sequencing (NGS) will add more substance to the existing approaches of phylogenetic studies. These approaches would be helpful not only in management of germplasm collections by avoiding redundancy but also in authentication of the genus Ocimum.

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43. Sarwat, M., Das, S., Srivastava, P. S., 2011a. A comparison of the AFLP and SAMPL molecular markers in characterizing genetic diversity of Terminalia arjuna- the backbone of tasar silk industry. Plant Syst. Evol. 293, 13-23. 44. Srivastava, S., Mishra, N., 2009. Genetic Markers- A Cutting Edge Technology In Herbal Drug Research. J. Chem. Pharm. Res. 1, 1-18. 45. Sarwat, M., Das, S., Srivastava, P.S., 2011b. Estimation of genetic diversity and evaluation of relatedness through molecular markers among medicinally important trees: Terminalia arjuna, T. chebula and T. bellerica. Mol Biol. Rep. 38, 50255036. 46. Bornet, B., Branchard, M., 2001. Nonanchored inter simple sequence repeat (ISSR) markers: reproducible and specific tools for genome fingerprinting. Plant Mol. Biol. Rep. 19, 209-215. 47. Lal, S., Mistry, K. N., Thaker, R., Shah, S. D., Vaidya, A. P. B., Patel, R., 2012. Genetic diversity assessment in six medicinally important species of Ocimum from Central Gujarat (India) utilizing RAPD, ISSR and SSR markers. Int. J. App. Biol. Res. 2, 279-288. 48. Li, G., Quiros, C. F., 2001. Sequence related amplified polymorphism (SRAP): a new marker system based on simple PCR reaction: its application to mapping and gene tagging in Brassica. Theor. Appl. Genet. 103, 455-461. 49. Agarwal, M., Shrivastava, N., Padh, H., 2008. Advances in molecular marker techniques and their applications in plant sciences. Plant Cell. Rep. 27, 617– 631. 50. Gulsen, O., Uzun, A., Canan, I., Seday, U., Canihos, E., 2010. A new citrus linkage map based on SRAP, SSR, ISSR, POGP, RGA, and RAPD markers. Euphytica 173, 265-277. 51. Chen, S. Y., Dai, T. X., Chang, Y. T., Wang, S. S., Ou, S. L., Chuang, W. L., Chuang, C. Y., Lin, Y. H., Lin, Y. Y., Ku, H. M., 2013. Genetic diversity among Ocimum species based on ISSR, RAPD, SRAP markers. Austr. J. Crop Sci. 7, 1463-1471. 52. Christina, V. L. P., Annamalai, A., 2014. Nucleotide based validation of Ocimum species by evaluating three candidate barcodes of the chloroplast region. Mol. Ecol. Resour. 14, 60-68. 53. Sarwat, M., Yamdagni, M. M., 2014. DNA barcoding, microarrays and next generation sequencing: recent tools for genetic diversity estimation and authentication of medicinal plants. Crit. Rev. Biotech. 29, 1-3. 54. Khare, C. P., 2007. Indian medicinal plant- an illustrated dictionary. Springer publication, pp. 445449. 55. Das, S. K., Vasudevan, D. M., 2006. Tulsi: The Indian holy power plant. Nat. Prod. Rad. 5, 279-283. 56. Singh, E., Sharma, S., Dwivedi, J., Sharma, S., 2012. Diversified potentials of Ocimum sanctum Linn (Tulsi): An exhaustive survey. J. Nat. Prod. Plant. Resour. 2, 39-48. 57. Vieira, R. F., Goldsbrough, P., Simon, J. E., 2003. Genetic diversity of Basil (Ocimum spp.) based on

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